Unit responses in the anterior and posterior hypothalamus to stimulation of the splanchnic and sciatic nerves and to photic stimulation

By extracellular recording of spike discharges the sensory properties of neurons of the anterior and posterior regions of the cat hypothalamus were studied during stimulation of the splanchnic and sciatic nerves and during photic stimulation. Hypothalamic neurons were shown to be characterized by wide convergence of heterosensory excitation: 68% of spontaneously active hypothalamic neurons responded to somatovisceral and photic stimulation. Some posterior hypothalamic neurons responded to somatovisceral stimulation but not to photic stimulation. Neurons responding only to photic stimulation were found in the anterior hypothalamus; no neurons responding only to visceral stimulation were found in the hypothalamus. Total convergence of somatic and visceral afferentation of neurons of the posterior and anterior hypothalamus was observed. Mostly responses of phasic type were obtained to stimulation of all modalities. The study of the quantitative ratio between responses of excitatory and inhibitory types showed that the former predominate. The principles governing the functional organization of hypothalamic afferent systems are discussed.Academician L. A. Orbeli Institute of Physiology, Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 8, No. 3, pp. 276–282, May–June, 1976.     

Chronic Functional Consequences of Adult Infraorbital Nerve Transection for Rat Trigeminal Subnucleus Interpolaris

In adult rats, transection of the infraorbital nerve and subsequent regeneration have been shown to result in altered somatotopic organization and changes in response properties of primary afferents within the trigeminal ganglion. The present study examined how these changes affect the postsynaptic targets of these neurons within subnucleus interpolaris of the trigeminal brainstem. Extra-cellular recordings were made from 330 cells in normal rats and 424 cells in rats surviving 57-290 days after transection of the infraorbital nerve in adulthood. Adult infraorbital nerve transection resulted in significant functional reorganization within subnucleus interpolaris. Relative to normal rats, the major changes can be summarized as follows: (1) a decrease in the dorsoventral extent of infraorbital representation; (2) a disruption of inter- and intradivisional somatotopic organization; (3) an increase in the proportion of cells with no discernible receptive field; (4) an increase in receptive field size for cells with infraorbital receptive field components; (5) the appearance of a significant proportion of cells with discontinuous receptive fields; (6) an increase in the proportion of cells exhibiting interdivisional convergence; (7) significant changes in the types of receptor surfaces activating local-circuit neurons with infraorbital receptive field components; (8) the appearance of a significant proportion of cells exhibiting convergence of different receptor surfaces; (9) significant changes in the dynamic response characteristics of cells with infraorbital receptive field components; and (10) an increase in the proportion of spontaneously active infraorbital-responsive cells. The changes observed were quite similar to those reported in adult subnucleus interpolaris following neonatal infraorbital nerve transection. The majority of changes observed in both studies can be most parsimoniously explained by alterations of primary afferents. However, central mechanisms may be more likely substrates for others. Regardless of the mechanism, the mature rodent trigeminal system appears capable of considerable functional reorganization following peripheral nerve damage.     

Chronic functional consequences of adult infraorbital nerve transection for rat trigeminal subnucleus interpolaris

In adult rats, transection of the infraorbital nerve and subsequent regeneration have been shown to result in altered somatotopic organization and changes in response properties of primary afferents within the trigeminal ganglion. The present study examined how these changes affect the postsynaptic targets of these neurons within subnucleus interpolaris of the trigeminal brainstem. Extracellular recordings were made from 330 cells in normal rats and 424 cells in rats surviving 57-290 days after transection of the infraorbital nerve in adulthood. Adult infraorbital nerve transection resulted in significant functional reorganization within subnucleus interpolaris. Relative to normal rats, the major changes can be summarized as follows: (1) a decrease in the dorsoventral extent of infraorbital representation; (2) a disruption of inter- and intradivisional somatotopic organization; (3) an increase in the proportion of cells with no discernible receptive field; (4) an increase in receptive field size for cells with infraorbital receptive field components; (5) the appearance of a significant proportion of cells with discontinuous receptive fields; (6) an increase in the proportion of cells exhibiting interdivisional convergence; (7) significant changes in the types of receptor surfaces activating local-circuit neurons with infraorbital receptive field components; (8) the appearance of a significant proportion of cells exhibiting convergence of different receptor surfaces; (9) significant changes in the dynamic response characteristics of cells with infraorbital receptive field components; and (10) an increase in the proportion of spontaneously active infraorbital-responsive cells. The changes observed were quite similar to those reported in adult subnucleus interpolaris following neonatal infraorbital nerve transection. The majority of changes observed in both studies can be most parsimoniously explained by alterations of primary afferents. However, central mechanisms may be more likely substrates for others. Regardless of the mechanism, the mature rodent trigeminal system appears capable of considerable functional reorganization following peripheral nerve damage.     

Modification of synapse formation of accessory olfactory bulb neurons by coculture with vomeronasal neurons

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.     

Ultramicroscopy Reveals Axonal Transport Impairments in Cortical Motor Neurons at Prion Disease

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.     

Cell structure and response properties in the trigeminal subnucleus oralis

Extra- and intracellular recording, electrical stimulation, receptive field mapping, and horseradish peroxidase injection techniques were used to study the structure of functionally identified neurons in trigeminal (V) brainstem subnucleus oralis of the rat. Of 15 heavily labeled cells located within oralis, 4 were local-circuit neurons with receptive fields restricted to either an incisor, guard hairs, one vibrissa, or deep facial tissue (nociceptors). Their morphologies were highly varied, with expansive and spiny dendritic trees and recurrent and intersubnuclear axon collaterals. Oralis local-circuit neurons therefore most closely resembled non-vibrissa-sensitive local-circuit cells in adjacent subnucleus interpolaris. Six other stained cells projected to contralateral thalamus, and two others projected to ipsilateral cerebellum. They typically had intramodality convergent receptive fields (i.e., spanning more than one receptor organ, such as multiple vibrissae or teeth) with widespread dendritic trees, and were therefore indistinguishable from similarly projecting cells in interpolaris. Two other cells projected to the ipsilateral spinal cord, as well as other V brainstem subnuclei. One of these responded to high-threshold mechanical stimulation of teeth; the other was discharged by deflection of one mystacial vibrissa. Their dendrites were very widespread and ended in spiny and bulbous appendages. Local axon collaterals were also extensive. The remaining oralis cell had two axons, one projecting to the thalamus, the other to the spinal cord. Its receptive field expressed convergence from multiple receptor organs, including vibrissae, guard hairs, and skin. Its somadendritic morphology was similar to that of oralis cells projecting only to thalamus. We conclude that, with some exceptions, the extensive dendritic trees, axon branching, convergence, and functional diversity of oralis cells approximate those described previously for functionally equivalent neurons in interpolaris (Jacquin et al., 1989a,b). Such anatomical and physiological properties are rarely seen, however, in nucleus principalis (Jacquin et al., 1988a). The structure and function of three atypical principalis cells with structural and functional characteristics typical of oralis cells are also described. It is argued that such cells are rostrally displaced oralis cells.     

Neuronal mechanisms of interaction of Deiters nucleus with the cerebral cortex.

The effects of stimulation of the vestibular nerve and five different cerebral cortex areas on the neuronal activity of the lateral vestibular nucleus of Deiters were studied. Stimulation of the cerebral cortex is shown to lead to antidromic and synaptic activation of Deiters neurons. The synaptic potentials of Deiters neurons evoked from the cerebral cortex were of mono- and polysynaptic origin. In particular, stimulation of the cerebral cortex evoked in Deiters neurons mono- and polysynaptic excitatory postsynaptic potentials. Collaterals of vestibulospinal neurons reaching different cortex fields as well as convergence of influences from these cortex fields on Deiters neurons were revealed. Inhibitory effects of the cerebral cortex on Deiters neurons were of polysynaptic origin and occurred rarely. The topical correlation between Deiters nucleus and different areas of the cerebral cortex was found. The peculiarities and functional significance of the effects obtained are discussed.     

Otolith and semicircular canal inputs to single vestibular neurons in cats.

Researchers studied the convergence of the vertical posterior semicircular canal (PC), saccular nerves (SAC), utricular nerves (UT), and horizontal semicircular canal nerves (HC) on single vestibular neurons. The vestibular neurons were categorized by their innervating targets. Vestibular neurons were classified as vestibulospinal proper neurons (VS), vestibulo-ocular proper neurons (VO), vestibulo-oculo-spinal neurons sending axon collaterals to the extraocular motoneuron pools and spinal cord (VOS), and vestibular nucleus neurons without axons to the oculomotor nuclei or the spinal cord (V). Results indicate that the percentage of convergence of VS neurons was higher that that of neurons sending axons to the oculomotor nuclei (VO and VOS). They conclude that the convergence of canal and otolith inputs likely contributes mainly to vestibulospinal reflexes by sending inputs to the neck and other muscles during head inclination, which creates the combined stimuli of angular and linear acceleration.     

Reprogramming of HUVECs into Induced Pluripotent Stem Cells (HiPSCs), Generation and Characterization of HiPSC-Derived Neurons and Astrocytes

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for “patient-matched” cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca²⁺ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.     

Neurons exclusively express N-Bak, a BH3 domain-only Bak isoform that promotes neuronal apoptosis

Bak is generally recognized as a multidomain, pro-apoptotic member of the Bcl-2 family. Bak and Bax are functionally redundant in non-neuronal cells and represent a mitochondrial convergence point for cell death signaling pathways. This functional redundancy, however, may not exist in neurons in which the single deletion of Bax is sufficient to confer protection against a variety of cytotoxic insults. In the present study, we demonstrate that postnatal cortical and cerebellar granule neurons exclusively express an alternatively spliced, BH3 domain-only form of Bak (N-Bak), whereas astrocytes express only the full-length, multidomain form. Overexpression of N-Bak promotes Bax translocation in HeLa cells and induces neuronal cell death in cortical, hippocampal, and cerebellar granule neurons in a Bax-dependent manner. N-Bak interacts with Bcl-XL but not BAX, suggesting an indirect mechanism for promoting Bax translocation to the mitochondria. N-Bak message and protein levels are elevated in cortical neurons in response to DNA damage, and subsequent induction of neuronal death is significantly delayed by expressing a full-length Bak antisense plasmid. These results demonstrate that postnatal neurons solely express a BH3 domain-only form of Bak, which contributes to DNA damage-induced neuronal apoptosis. The absence of full-length Bak expression explains the near exclusive requirement for Bax in neuronal apoptosis.     

Sources of thalamic projections in the region of vibrissal representation in the rat somatosensory cortex studied by horseradish peroxide labeling

The retrograde horseradish peroxidase (HRP) transport method was used to study the location and morphology of neuron groups in the ventrobasal complex of the thalamus projecting to the region of vibrissal representation in the somatosensory cortex in rats. Injection of HRP into a circumscribed region of the somatosensory cortex revealed the following pattern of organization of the thalamocortical relay groups of neurons. Labeled neurons were located in the ventroposterolateral nucleus of the ventrobasal complex and were associated in groups 100–120 µ in diameter. Staining of several groups, even after minimal injections of HRP, and an increase in the number of labeled cells in each group with an increase in the zone of injection of HRP in the cortex suggest the presence of both convergence and divergence of specific thalamocortical pathways. The different shapes of the relay neurons and differences in the degree of HRP accumulation by them may indicate differences in their functional role in thalamocortical integration.Research Institute of Neurocybernetics, Rostov State University. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 631–635, November–December, 1982.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

果蝇幼虫的视觉系统

视觉对于动物的生存和行为来说是非常重要的。虽然果蝇幼虫的视觉神经系统在组织结构上比成虫简单,但是也具有一定的复杂性和多样性。在最近几年中,随着对果蝇幼虫视觉系统功能的研究取得重要进展,我们对于果蝇幼虫视觉系统的认识更加丰富了。果蝇幼虫视觉系统的结构中,最初级的光感受神经元包括三类,一类是BO/BN(Bolwig's organ/Bolwig's nerve),一类是表达感光分子CRY(cryptochrome)的神经元,另外一类是Ⅳ型DA(classⅣdendriticarborization)神经元;果蝇幼虫视觉系统的次级神经元主要是光节律相关的侧神经元(lateralneurons,LN),它表达Per(period)、Tim(timeless)及Pdf(pigment dispersion factor)等节律相关的蛋白分子;而第三级神经元包括更为下游的、表达果蝇促胸腺激素且直接调控幼虫光偏好的NP394神经元。这三级神经元构成了我们现在所了解的果蝇幼虫视觉神经系统的基本框架。     

Semaphorin-1a controls receptor neuron-specific axonal convergence in the primary olfactory center of Drosophila

In the olfactory system of Drosophila, 50 functional classes of sensory receptor neurons (ORNs) project in a highly organized fashion into the CNS, where they sort out from one another and converge into distinct synaptic glomeruli. We identified the transmembrane molecule Semaphorin-1a (Sema-1a) as an essential component to ensure glomerulus-specific axon segregation. Removal of sema-1a in ORNs does not affect the pathfinding toward their target area but disrupts local axonal convergence into a single glomerulus, resulting in two distinct targeting phenotypes: axons either intermingle with adjacent ORN classes or segregate according to their odorant receptor identity into ectopic sites. Differential Sema-1a expression can be detected among neighboring glomeruli, and mosaic analyses show that sema-1a functions nonautonomously in ORN axon sorting. These findings provide insights into the mechanism by which afferent interactions lead to synaptic specificity in the olfactory system.     

Temporal course of disinhibition of the visual cortex neurons and their sensitivity to cross-like figures

In acute experiments with narcotized and paralyzed cats, we studied responses of 74 striate neurons to cross-like figure under synchronous and asynchronous presentation of its lines. The aim of the study was to characterize the temporal course of interaction between three RF zones: main excitatory, end-inhibitory, and side disinhibitory ones. Previously we have found that this interaction is responsible for sensitivity to a cross in near 3/4 of cat striate cells with such sensitivity. In neurons with sensitivity to a cross, we found two types of temporal interaction between zones of RF. In the 1st type cells (14/23), the response significantly increased if the disinhibitory and the main excitatory zones of RF were stimulated simultaneously. Neurons of the 2nd type (9/23) revealed opposite temporal function: synchronous activation of RF zones evoked a minimal response. Simulation shows that the 1st type of behavior is connected with disinhibitory mechanism, while that of the 2nd type--with combination of this mechanism with convergence of orientation detectors of the previous functional level.     

乙酰胆碱,谷氨酸与GABA对丘脑腹内侧核神经元活动的影响

本文采用微电泳方法观察到在大鼠丘脑腹内侧核（ＶＭ）,微电泳给予乙酰胆碱（ＡＣＨ）使所有受试神经元自发放电频率加快,谷氨酸（ＧＬＵ）使大多数神经元放电加快,它们的作用依赖于电流强度;而γ氨基丁酸（ＧＡＢＡ）和氯苯氨丁酸则抑制大多数神经元的放电活动,但前者的作用快速而暂短,而后者的作用相对缓慢而持久。在微电泳ＡＣＨ或ＧＬＵ的过程中,给予ＧＡＢＡ可拮抗它们的兴奋作用。双钴碱使大多数神经元的自发放电频率加快,而阿托品和ＭＫ８０１对自发放电的影响较小。这些结果表明ＧＡＢＡ,ＡＣＨ和ＧＬＵ等递质活动在同一ＶＭ神经元有重要的会聚作用;ＧＡＢＡ对ＶＭ神经元有紧张性抑制作用。     

The organization of plurisegmental mechanosensitive interneurons in the central nervous system of the wandering spider Cupiennius salei

Summary In spiders the bulk of the central nervous system (CNS) consists of fused segmental ganglia traversed by longitudinal tracts, which have precise relationships with sensory neuropils and which contain the fibers of large plurisegmental interneurons. The responses of these interneurons to various mechanical stimuli were studied electrophysiologically, and their unilateral or bilateral structure was revealed by intracellular staining. Unilateral interneurons visit all the neuromeres on one side of the CNS. They receive mechanosensory input either from a single leg or from all ipsilateral legs via sensory neurons that invade leg neuromeres and project into specific longitudinal tracts. The anatomical organization of unilateral interneurons suggests that their axons impart their information to all ipsilateral leg neuromeres. Bilateral interneurons are of two kinds, symmetric and asymmetric neurons. The latter respond to stimulation of all legs on one side of the body, having their dendrites amongst sensory tracts of the same side of the CNS. Anatomical evidence suggests that their terminals invade all four contralateral leg neuromeres. Bilaterally symmetrical plurisegmental interneurons have dendritic arborizations in both halves of the fused ventral ganglia. They respond to the stimulation of any of the 8 legs. A third class of cells, the ascending neurons have unilateral or bilateral dendritic arborizations in the fused ventral ganglia and show blebbed axons in postero-ventral regions of the brain. Their response characteristics are similar to those of other plurisegmental interneurons. Descending neurons have opposite structural polarity, arising in the brain and terminating in segmental regions of the fused ventral ganglia. Descending neurons show strong responses to visual stimulation. Approximately 50% of all the recorded neurons respond exclusively to stimulation of a single type of mechanoreceptor (either tactile hairs, or trichobothria, or slit sensilla), while the rest respond to stimulation of a variety of sensilla. However, these functional differences are not obviously reflected by the anatomy. The functional significance of plurisegmental interneurons is discussed with respect to sensory convergence and the coordination of motor output to the legs. A comparison between the response properties of certain plurisegmental interneurons and their parent longitudinal tracts suggests that the tracts themselves do not reflect a modality-specific organization.Abbreviations BPI bilateral plurisegmental interneuron - CNS central nervous system - FVG fused ventral ganglia - LT longitudinal tract - PI plurisegmental interneuron - PSTH peristimulus timehistogram - UPI unilateral plurisegmental interneuron     

Experimental study of early olfactory neuron differentiation and nerve formation using quail-chick chimeras

For the formation of a functional olfactory system, the key processes are neuronal differentiation, including the expression of one or the other olfactory receptors, the correct formation of the nerve and organization of periphero-central connections. These processes take place during embryonic development starting from early stages. Consequently, avian embryos afford an attractive model to study these mechanisms. Taking advantage of species-specific equipment of olfactory receptors genes in different bird species, interspecific avian chimeras were set up by grafting early chick olfactory placodes in same stage quail embryos. Their analysis was performed using different complementary approaches. In situ hybridisation using probes to different chick olfactory receptor (COR) genes indicated that the choice of expression of an olfactory receptor by a neuron is independent of the environment of the olfactory placode and of interactions with the central nervous system. Futhermore, a chick olfactory receptor gene subgroup (COR3 ), absent in the host genome, was expressed by neurons from the graft. The question was then raised of the consequences of such heterospecific differentiation on axonal projections and fiber convergence. The DiI labeling of olfactory fibres in chimeras revealed anomalies in the formation of the nerve from the chick graft. In agreement with the hypothesis of olfactory receptor (OR) involvement in axonal guidance and periphero-central synapse organisation, the presence of migrating cells and axonal fibres from the graft, expressing foreign ORs and having different interactions with the host environment than the host fibres and migrating cells, might explain these anomalies.     

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.